The two hallmarks of hepatocarcinogenesis associated with the c-myc/TGF-a are widespread dysplasia and profound chromosomal abnormalities. However, the most remarkable biological consequence of constitutive co-expression of c-myc and TGF-a transgenes is severe DNA damage. In 2-month-old double transgenic hepatocytes, the frequency of chromosomal breakage was increased nearly tenfold, whereas the number of aberrations observed in c-myc and TGF-a single transgenic hepatocytes was only 1.3- and 3-times background, respectively. Moreover, the presence of nonrandom chromosomal breaks recorded prior to tumor development and persistent enhancement of chromosomal damage in hepatocellular carcinomas suggested that TGF-a/c-myc hepatocytes exhibited a mutator phenotype. In light of recent evidence indicating that ligand binding to cell surface receptors linked to tyrosine kinase activity can trigger signal transduction pathways leading to production of intracellular reactive oxygen species (ROS), and given the known carcinogenic properties of ROS, we hypothesized that enhanced metabolic generation of oxygen radicals in rapidly proliferating c-myc/TGF-a transgenic hepatocytes might be responsible for genetic instability and acceleration of liver cancer in this animal model. By two months of age, production of ROS is significantly elevated in TGF-a/c-myc transgenic hepatocytes versus either wild type or c-myc single transgenic cells, and occurred in parallel with an increase in lipid peroxidation. Concomitantly with a rise in oxidant levels, antioxidant defenses were decreased, including total glutathione content and the activity of glutathione peroxidase. Furthermore, specific deletions were detected in mtDNA as early as five weeks of age in the transgenic mice. We conclude from these data that co-expression of TGF-a and c-myc transgenes in mouse liver promotes overproduction of ROS and thus creates an oxidative stress environment. This phenomenon may account for the massive DNA damage and acceleration of hepatocarcinogenesis observed in the TGF-a/c-myc mouse model.